In order to generate sufficient signal for analysis, many applications in genomics and biomedical research utilize the conversion of nucleic acid molecules in a library into separate, or separable, libraries of amplicons of the molecules, e.g. Margulies et al, Nature 437: 376-380 (2005); Mitra et al, Nucleic Acids Research, 27: e34 (1999); Shendure et al, Science, 309: 1728-1732 (2005); Brenner et al, Proc. Natl. Acad. Sci., 97: 1665-1670 (2000); and the like. Several techniques have been used for making such conversions, including hybrid selection (e.g., Brenner et al, cited above); in-gel polymerase chain reaction (PCR) (e.g. Mitra et al, cited above); bridge amplification (e.g. Shapero et al, Genome Research, 11: 1926-1934 (2001)); and emulsion PCR (emPCR) (e.g. Margulies et al, cited above). Most of these techniques employ particulate supports, such as beads, which spatially concentrate the amplicons for enhanced signal-to-noise ratios, as well as other benefits, such as, better reagent access.
These techniques have several drawbacks. In some cases, amplicons are either in a planar format (e.g. Mitra et al, cited above; Adessi et al, Nucleic Acids Research, 28: e87 (2000)), which limits ease of manipulation or reagent access, or the amplicons are on bead surfaces, which lack sufficient fragment density or concentration for adequate signal-to-noise ratios. Another disadvantage of traditional beads is their solid structure and thus lack of diffusivity. Even “macroporous” particles are solid structures that do not diffuse analytes and reporter molecules as quickly or efficiently as gel-based materials. The gel particles have the advantage of increasing diffusivity of analytes and reporter molecules through the gel-based material. This increases the efficiency of moving/diffusing analytes and reporter molecules, such as, for example, nucleotides being washed over the sensory chip in an apparatus. For example, hydrogen ions and DNA can diffuse through a gel bead with great efficiency regardless of where they are produced and enter the bead because of the gel material. It would also be useful if supports were available that were capable of providing a higher density of analyte binding or attachment sites, particularly for clonal populations of nucleic acid fragments, thereby allowing production of supports with higher template loads.
Gels have been widely used as supports in analytical and synthetic processes and as encapsulating agents, e.g. Weaver et al, U.S. Pat. No. 5,055,390; Tmovsky et al, U.S. Pat. No. 6,586,176, and have interiors accessible to analytical reagents. However, such particulates are limited in that they are typically produced with widely varying size distributions, particularly at lower size ranges, e.g. less than about 30 μm, which makes them unsuitable for many exacting analytical applications, such as large scale DNA sequencing.
U.S. Patent Application Publication No. 2010/0304982, however, describes methods and compositions for making porous microparticles or polymer particles, which may be readily loaded with analytes, such as amplicons of nucleic acid fragments.
There exists a desire, however, for methods of making polymer particles and scaffold nucleic acid polymeric particles (SNAPPs) that are time or cost efficient and also for methods that are capable of producing a high yield of particles, which may also be of high or consistent quality. There also exists a desire for methods of making SNAPPs of high sequencing quality. The inventors have now discovered such novel methods of making polymer particles and SNAPPs and the products produced therefrom.
In the following description, various aspects and embodiments of the invention will become evident. In its broadest sense, the invention could be practiced without having one or more features of these aspects and embodiments. Further, these aspects and embodiments are exemplary. Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practicing the invention. The objects and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.